Birunthi Niranjan

Estrogen receptor-beta activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNFalpha mediated

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor–β (ERβ) in BPH and PCa. ERβ agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNFα knock-out mice fail to respond to ERβ agonist, demonstrating the requirement for TNFα signaling. In human tissues, ERβ agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the CD133+ enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ERβ causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ERβ agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ERβ agonist for treatment of PCa and/or BPH with or without androgen withdrawal.

Activin C Antagonizes Activin A in Vitro and Overexpression Leads to Pathologies in Vivo

Activin A is a potent growth and differentiation factor whose synthesis and bioactivity are tightly regulated. Both follistatin binding and inhibin subunit heterodimerization block access to the activin receptor and/or receptor activation. We postulated that the activin-beta(C) subunit provides another mechanism regulating activin bioactivity. To test our hypothesis, we examined the biological effects of activin C and produced mice that overexpress activin-beta(C). Activin C reduced activin A bioactivity in vitro; in LNCaP cells, activin C abrogated both activin A-induced Smad signaling and growth inhibition, and in LbetaT2 cells, activin C antagonized activin A-mediated activity of an follicle-stimulating hormone-beta promoter. Transgenic mice that overexpress activin-betaC exhibited disease in testis, liver, and prostate. Male infertility was caused by both reduced sperm production and impaired sperm motility. The livers of the transgenic mice were enlarged because of an imbalance between hepatocyte proliferation and apoptosis. Transgenic prostates showed evidence of hypertrophy and epithelial cell hyperplasia. Additionally, there was decreased evidence of nuclear Smad-2 localization in the testis, liver, and prostate, indicating that overexpression of activin-beta(C) antagonized Smad signaling in vivo. Underlying the significance of these findings, human testis, liver, and prostate cancers expressed increased activin-betaC immunoreactivity. This study provides evidence that activin-beta(C) is an antagonist of activin A and supplies an impetus to examine its role in development and disease.

A preclinical xenograft model of prostate cancer using human tumors

Most cases of prostate cancer are now diagnosed as moderate-grade localized disease. These tumor specimens are important tools in the discovery and translation of prostate cancer research; however, unlike more advanced tumors, they are notoriously difficult to grow in the laboratory. We developed a system for efficiently xenografting localized human prostate cancer tissue, and we adapted this protocol to study the interactions between the specific subsets of epithelial and stromal cells. Fresh prostate tissues or isolated epithelial cells are recombined with mouse seminal vesicle mesenchyme (SVM) and grafted under the renal capsule of immunodeficient mice for optimum growth and survival. Alternatively, mouse mesenchyme can be replaced with human prostate fibroblasts in order to determine their contribution to tumor progression. Grafts can be grown for several months to determine the effectiveness of novel therapeutic compounds when administered to host mice, thereby paving the way for personalizing the treatment of individual prostate cancers.

A bioengineered microenvironment to quantitatively measure the tumorigenic properties of cancer-associated fibroblasts in human prostate cancer

Stromal-epithelial cell interactions play an important role in cancer and the tumor stroma is regarded as a therapeutic target. In vivo xenografting is commonly used to study cellular interactions not mimicked or quantified in conventional 2D culture systems. To interrogate the effects of tumor stroma (cancer-associated fibroblasts or CAFs) on epithelia, we created a bioengineered microenvironment using human prostatic tissues. Patient-matched CAFs and non-malignant prostatic fibroblasts (NPFs) from men with moderate (Gleason 7) and aggressive (Gleason 8-9 or castrate-resistant) prostate cancer were cultured with non-tumorigenic BPH-1 epithelial cells. Changes in the morphology, motility and phenotype of BPH-1 cells in response to CAFs and NPFs were analyzed using immunofluorescence and quantitative cell morphometric analyses. The matrix protein gene expression of CAFs, with proven tumorigenicity in vivo, had a significantly different gene expression profile of matrix proteins compared to patient matched NPFs. In co-culture with CAFs (but not NPFs), BPH-1 cells had a more invasive, elongated phenotype with increased motility and a more directed pattern of cell migration. CAFs from more aggressive tumors (Gleason 8-9 or CRPC) were not quantitatively different to moderate grade CAFs. Overall, our bioengineered microenvironment provides a novel 3D in vitro platform to systematically investigate the effects of tumor stroma on prostate cancer progression.

Regulation of the transcriptional coactivator FHL2 licenses activation of the androgen receptor in castrate-resistant prostate cancer

It is now clear that progression from localized prostate cancer to incurable castrate-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR), signaling independently of androgen. Thus, there remains a strong rationale to suppress AR activity as the single most important therapeutic goal in CRPC treatment. Although the expression of ligand-independent AR splice variants confers resistance to AR-targeted therapy and progression to lethal castrate-resistant cancer, the molecular regulators of AR activity in CRPC remain unclear, in particular those pathways that potentiate the function of mutant AR in CRPC. Here, we identify FHL2 as a novel coactivator of ligand-independent AR variants that are important in CRPC. We show that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathway that shows differential activation in prostate epithelial versus prostate cancer cell lines. We further identify a novel FHL2-AR-filamin transcription complex, revealing how deregulation of this axis promotes the constitutive, ligand-independent activation of AR variants, which are present in CRPC. Critically, the calpain-cleaved filamin fragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostate cancer. Thus, our work provides mechanistic insight into the enhanced AR activation, most notably of the recently identified AR variants, including AR-V7 that drives CRPC progression. Furthermore, our results identify the first disease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. These results offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other human cancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer.

Characterization of gametogenetin 1 (GGN1) and its potential role in male fertility through the interaction with the ion channel regulator, cysteine-rich secretory protein 2 (CRISP2) in the sperm tail

Cysteine-rich secretory protein 2 (CRISP2) is a testis-enriched protein localized to the sperm acrosome and tail. CRISP2 has been proposed to play a critical role in spermatogenesis and male fertility, although the precise function(s) of CRISP2 remains to be determined. Recent data have shown that the CRISP domain of the mouse CRISP2 has the ability to regulate Ca(2+) flow through ryanodine receptors (RyR) and to bind to MAP kinase kinase kinase 11 (MAP3K11). To further define the biochemical pathways within which CRISP2 is involved, we screened an adult mouse testis cDNA library using a yeast two-hybrid assay to identify CRISP2 interacting partners. One of the most frequently identified CRISP2-binding proteins was gametogenetin 1 (GGN1). Interactions occur between the ion channel regulatory region within the CRISP2 CRISP domain and the carboxyl-most 158 amino acids of GGN1. CRISP2 does not bind to the GGN2 or GGN3 isoforms. Furthermore, we showed that Ggn1 is a testis-enriched mRNA and the protein first appeared in late pachytene spermatocytes and was up-regulated in round spermatids before being incorporated into the principal piece of the sperm tail where it co-localized with CRISP2. These data along with data on RyR and MAP3K11 binding define the CRISP2 CRISP domain as a protein interaction motif and suggest a role for the GGN1-CRISP2 complex in sperm tail development and/or motility.

A comparative assessment of α-lipoic acid N-phenylamides as non-steroidal androgen receptor antagonists both on and off gold nanoparticles

A group of α-lipoic acid N-phenylamides were synthesized employing a variety of amide coupling protocols utilizing electron deficient anilines. These compounds were then assessed for their ability to block androgen-stimulated proliferation of a human prostate cancer cell line, LNCaP. These structurally simple compounds displayed anti-proliferative activities at, typically, 5-20 μM concentrations and were comparable to a commonly used anti-androgen Bicalutamide®. The inclusion of a disulfide (RS-SR) moiety, serving as an anchor to several metal nanoparticle systems (Au, Ag, Fe(2)O(3), etc.), does not impede any biological activity. Conjugation of these compounds to a gold nanoparticle surface resulted in a high degree of cellular toxicity, attributed to the absence of a biocompatible group such as PEG within the organic scaffold.

Primary Culture and Propagation of Human Prostate Epithelial Cells

Basic and translational (or preclinical) prostate cancer research has traditionally been conducted with a limited repertoire of immortalized cell lines, which have homogeneous phenotypes and have adapted to long-term tissue culture. Primary cell culture provides a model system that allows a broader spectrum of cell types from a greater number of patients to be studied, in the absence of artificially induced genetic mutations. Nevertheless, primary prostate epithelial cell culture can be technically challenging, even for laboratories experienced in immortalized cell culture. Therefore, we provide methods to isolate and culture primary epithelial cells directly from human prostate tissue. Initially, we describe the isolation of bulk epithelial cells from benign or tumor tissues. These cells have a predominantly basal/intermediate phenotype and co-express cytokeratin 8/18 and high molecular weight cytokeratins. Since prostatic stem cells play a major role in disease progression and are considered to be a therapeutic target, we also describe a prospective approach to specifically isolate prostatic basal cells that include both stem and transit-amplifying basal populations, which can be studied independently or subsequently differentiated to supply luminal cells. This approach allows the study of stem cells for the development of new therapeutics for prostate cancer.

Preliminary investigations into triazole derived androgen receptor antagonists

A range of 1,4-substituted-1,2,3-N-phenyltriazoles were synthesized and evaluated as non-steroidal androgen receptor (AR) antagonists. The motivation for this study was to replace the N-phenyl amide portion of small molecule antiandrogens with a 1,2,3-triazole and determine effects, if any, on biological activity. The synthetic methodology presented herein is robust, high yielding and extremely rapid. Using this methodology a series of 17 N-aryl triazoles were synthesized from commercially available starting materials in less than 3h. After preliminary biological screening at 20 and 40 μM, the most promising three compounds were found to display IC50 values of 40-50 μM against androgen dependent (LNCaP) cells and serve as a starting point for further structure-activity investigations. All compounds in this work were the focus of an in silico study to dock the compounds into the human androgen receptor ligand binding domain (hARLBD) and compare their predicted binding affinity with known antiandrogens. A comparison of receptor-ligand interactions for the wild type and T877A mutant AR revealed two novel polar interactions. One with Q738 of the wild type site and the second with the mutated A877 residue.

Synthesis and preliminary investigations into novel 1,2,3-triazole-derived androgen receptor antagonists inspired by bicalutamide.

A versatile and high yielding synthesis of novel androgen receptor (AR) antagonists is presented. Using this methodology, six 1,4-substituted-1,2,3-triazole derived bicalutamide mimics were synthesised in five steps and in isolated overall yields from 41% to 85%. Evaluation of these compounds for their anti-proliferative properties against androgen dependent (LNCaP) and independent (PC-3) cells showed promising IC50 values of 34-45 μM and 29-151 μM, respectively. The data suggest that the latter compounds may be an excellent starting point for the development of prostate cancer therapeutics for both androgen dependent and independent forms of this disease. Docking of these compounds (each enantiomer) in silico into the T877A mutated androgen receptor, as possessed by LNCaP cells, was also undertaken.